pS129 alpha-synuclein Western blot

Add 100µL of lysis buffer to the cell pellet and mix thoroughly by pipetting up and down until the cell pellet is completely resolved into a homogeneous solution.

Adjust the lysis buffer volume to the cell pellet size, keep the sample at Room temperature as soon as the lysis buffer has been added, and work quickly to prevent proteolysis.

Starting with a frozen cell pellet, keep it on dry ice initially and thaw it on regular ice as soon as everything is set up.

If the cell pellet is collected right before conducting the Western blot, keep it On ice until the lysis buffer is added.

Vortex and heat samples for 0h 10m 0s at 100°C.

Spin down at maximal speed for 0h 10m 0s. Transfer the supernatant to a new Eppendorf tube and discard the cell pellet.

Store the supernatant at -20°C in order to proceed with the protocol at a later point or as a positive control for later experiments.

Quantify protein concentration through a classical BCA assay (or other available techniques).

For each sample, mix an appropriate amount of protein with NuPAGE® LDS Sample Buffer (4x), reducing agent (10x) and sterile H2O for protein linearization and monomerization.

Load no more than 40µg protein per well (e.g. 30µg).

Heat samples for 0h 10m 0s at 95°C.

Prepare 500mL of running buffer 1X by mixing 475mL diH20 with 25mL NuPAGE® MES SDS Running Buffer (20x).

Place a 4-12 % Bis-Tris Gel in the gel electrophoresis chamber (after removing the tape and the comb and after rinsing) and fill the chamber with running buffer.

Use gel-loading tips to load the samples and 4µL of the protein ladder (Precision Plus Protein™ Dual Color Standards) onto the gel.

Run the gel at 150 V for approximately 0h 30m 0s – 0h 55m 0s until the protein ladder.

Use the iBlot Gel Transfer Device and the iBlot PVDF Transfer Stacks for dry blotting of proteins.

Boot the gel transfer device, select program “P0 7 min” (20v 1:00; 23v 4:00; 25v 2:00) and place the iBlot

anode stack (“bottom”), with the PVDF transfer membrane on top, into the gel transfer device.

Separate the two plastic plates of the gel cassette using a gel knife. Cut off excessive parts of the gel. Transfer the gel onto the PVDF membrane. Gently remove any air bubbles using a wetted blotting roller.

Wet a Whatman paper in diH2O and place it on top of the gel. Gently remove any air bubbles using the wetted blotting roller.

Place the iBlot cathode stack (“top”) on top and even the layers with the blotting roller. Place the iBlot sponge on the inner side of the iBlot lid in the right orientation.

Close the iBlot gel transfer device and start the program.

Fix membrane in 4% paraformaldehyde in PBS for 0h 40m 0s rocking at 95Room temperature.

Wash 3X with TBS (use and discard paraformaldehyde in compliance with the laboratory safety regulations).

Wash the PVDF membrane by gently rocking it in TBS for 0h 5m 0s.

When transferring the PVDF membrane or exchanging the solution in the blot box, make sure that the PVDF membrane never dries out.

Remove the TBS and add a sufficient volume of one part Odyssey blocking buffer (TBS) mixed with one part of TBS (1:1). Incubate for 1h 0m 0s on a rocker.

Exchange the blocking solution with the primary antibody solution, i.e. one part Odyssey blocking buffer (TBS) mixed with one part TBS + the primary antibodies at the desired dilution.

Incubate at 4°C on a rocker 1h 0m 0s.

Wash the PVDF membrane with TBS + 0.01% Tween20 on a rocker 3 times for 0h 10m 0s.

Exchange with the secondary antibody solution, i.e. one part Odyssey blocking buffer (TBS) mixed with one part TBS (1:1) + the secondary antibody at the desired dilution.

Incubate on a rocker for 2h 0m 0s at 4Room temperature protected from light, i.e. wrap the blot box in aluminum foil.

Wash the PVDF membrane with TBS + 0.01% Tween20 on a rocker 3 times for 0h 10m 0s protected from light.

Wash the PVDF membrane again with TBS until Tween-20 has been entirely removed and scan the blot using the Odyssey CLx Infrared Imager; for bands quantification use the Odyssey Software version 3.0.